Data:
High-throughput experiment
Thousands of SarsCov2 peptides binding to T-cells
TCR-dependent or TCR-independent (i.e.: KIR-mediated)?
3 cohorts:
AP: Leukemia patients who have had COVID-19 (n = 12)
BS: Healthy nurses who have had COVID-19 (n = 17)
BC: Healthy donors (n = 15)
Methods:
DNA-barcoding: Tagging each peptide-HLA pair uniquely
Flow cytometry: Sorting T-cells based on TCR vs KIR binding
| AP (N=13465) |
BS (N=7840) |
BC (N=10491) |
Overall (N=31796) |
|
|---|---|---|---|---|
| KIR_blocking | ||||
| 1 | 7541 (56.0%) | 4019 (51.3%) | 7877 (75.1%) | 19437 (61.1%) |
| 0 | 5924 (44.0%) | 3821 (48.7%) | 2614 (24.9%) | 12359 (38.9%) |
| log_fold_change | ||||
| Mean (SD) | -1.15 (3.29) | -2.25 (3.95) | -1.49 (3.69) | -1.53 (3.62) |
| Median [Min, Max] | -0.182 [-13.4, 9.71] | -0.508 [-12.0, 8.64] | -0.313 [-12.4, 11.2] | -0.290 [-13.4, 11.2] |
| protein | ||||
| ORF1 | 7239 (53.8%) | 4447 (56.7%) | 6103 (58.2%) | 17789 (55.9%) |
| Nucleocapsid | 707 (5.3%) | 434 (5.5%) | 548 (5.2%) | 1689 (5.3%) |
| Spike | 2619 (19.5%) | 1581 (20.2%) | 2062 (19.7%) | 6262 (19.7%) |
| ORF6 | 68 (0.5%) | 42 (0.5%) | 65 (0.6%) | 175 (0.6%) |
| Membrane | 785 (5.8%) | 465 (5.9%) | 545 (5.2%) | 1795 (5.6%) |
| ORF3 | 540 (4.0%) | 345 (4.4%) | 467 (4.5%) | 1352 (4.3%) |
| ORF7 | 288 (2.1%) | 175 (2.2%) | 227 (2.2%) | 690 (2.2%) |
| Envelope | 119 (0.9%) | 73 (0.9%) | 91 (0.9%) | 283 (0.9%) |
| ORF10 | 119 (0.9%) | 73 (0.9%) | 91 (0.9%) | 283 (0.9%) |
| ORF8 | 322 (2.4%) | 205 (2.6%) | 292 (2.8%) | 819 (2.6%) |
| Unknown | 659 (4.9%) | 0 (0%) | 0 (0%) | 659 (2.1%) |
| HLA | ||||
| HLA-C04:01 | 300 (2.2%) | 600 (7.7%) | 2400 (22.9%) | 3300 (10.4%) |
| HLA-C06:02 | 2695 (20.0%) | 1086 (13.9%) | 1164 (11.1%) | 4945 (15.6%) |
| HLA-C07:01 | 4014 (29.8%) | 2950 (37.6%) | 2655 (25.3%) | 9619 (30.3%) |
| HLA-C07:02 | 6456 (47.9%) | 3204 (40.9%) | 4272 (40.7%) | 13932 (43.8%) |
~ 32 000 bar-codes (patient-HLA-peptide combinations)
44 patients
4 HLA types
11 protein types
Flow cytometry data
56 patients - not total overlap with Barracoda
Not all patients have been tested with and without KIR-blocking
| KIR-blocked (N=30) |
Not KIR-blocked (N=26) |
Overall (N=56) |
|
|---|---|---|---|
| APC_positive | |||
| Mean (SD) | 0.682 (0.626) | 1.43 (2.58) | 1.03 (1.84) |
| Median [Min, Max] | 0.450 [0, 2.20] | 0.305 [0, 9.19] | 0.360 [0, 9.19] |
| DP_positive | |||
| Mean (SD) | 0.210 (0.376) | 0.0319 (0.0905) | 0.128 (0.294) |
| Median [Min, Max] | 0.0510 [0, 1.61] | 0 [0, 0.350] | 0.0105 [0, 1.61] |
| PE_positive | |||
| Mean (SD) | 2.86 (3.18) | 6.32 (9.09) | 4.46 (6.78) |
| Median [Min, Max] | 2.18 [0.100, 17.2] | 3.24 [0.160, 46.1] | 2.44 [0.100, 46.1] |
2 Fluorochromes:
PE: binding to SarsCov2
APC: binding to control peptides
DP = Double positive for APC and PE
The number of observations reduced drastically during wrangling:
Original: 31796
w. Flowjo: 22819
Significant: 542
Paired data to compare TCR-dependent and TCR-independent responses:
This further reduced the dataset size:
Original: 31796
Significant: 542
Paired: 22
11 pairs
1 normal
1 KIR-blocked
\[ Est. Freq = \left( \frac{count_{barcode}}{count_{total}} \cdot 100 \% \right) \cdot PE \]
\[ Est. Freq = \left( \frac{count_{barcode}}{count_{total}} \cdot 100 \% \right) \cdot PE \]
The paired dataset was really small, hard to draw reliable conclusions
New info from this research group indicates that the KIR-antibody used in this experiment only blocked KIR-C1 but not -C2
C0401 and C0601 are C2 ligands and were therefore not blocked
This would explain the unreliable results, and variation between cohorts
R4BDS - Group 25